Femtosecond to Millisecond Photo-dynamics of Third Generation Fluorescent Proteins

几个世纪以来，光学显微镜一直是生物学的核心。近几十年来，荧光显微镜已经发展成为一种非常敏感和普遍使用的生命科学工具。在20世纪90年代，由于绿色荧光蛋白（GFP）的发现，它的力量得到了极大的增强。这使得荧光标记（GFP）可以不可逆地、选择性地附着在特定的靶蛋白上表达，从而可以在活细胞中探测该蛋白的位置、动态和功能。最近，光开关荧光蛋白被发现。这些荧光蛋白可以通过光在荧光（开）和非荧光（关）状态之间可逆地切换。这一特性使得超分辨率荧光生物成像得以发展，将荧光显微镜的空间分辨率提高了10倍以上，从而可以在生命系统中观察到比光波长小得多的细节。然而，可光开关蛋白质的应用受到限制，因为用于从它们的“开”状态产生荧光的光也会使蛋白质“关”，导致图像褪色。最近发现的第三代荧光蛋白（3G FPs）提供了更高水平的控制，它具有三种状态，一个关闭状态和一个热平衡状态的开关状态。这三个状态可以被独立激发，因此关闭与观察和导通状态解耦。这一发展将导致增强的超分辨率成像，并具有开发新的多色成像方法的潜力。然而，连接这些状态的机制是完全未知的。我们的实验将使我们能够详细地探测光对3G FPs的所有三种状态的影响。我们将测量这三种状态之间相互转换的速率以及伴随它们的结构变化。由于最快的相互转化反应非常快（十亿分之一秒），我们将使用超快激光光谱学工具进行观测。我们的超快实验通过吸收光谱来探测反应物、中间体和产物态的居群，同时通过超快振动光谱来探测它们的结构。这些测量将导致3G FPs中光转换机制的详细图像。一旦机制建立，我们将应用化学生物学的工具在关键残基上进行突变，这将(i)测试我们对机制的想法，（ii）优化光开关速率，从而为生物成像产生卓越的3G FPs。光活性蛋白（如FPs）为实时观察蛋白质结构演变提供了独特的机会。特别是时间分辨振动光谱产生从飞秒到毫秒的结构数据。这一信息非常重要，因为它可以与蛋白质结构动力学的计算计算和独立实验结果进行比较。总之，这将产生最详细的洞察到蛋白质的动态经历其功能，这反过来又增强了我们对药物-蛋白质相互作用的本质的理解。

Optical microscopy has been central to biology for centuries. In recent decades fluorescence microscopy has developed as an exceptionally sensitive and universally employed tool in the life sciences. Its power was dramatically enhanced in the 1990s through the discovery of the green fluorescent protein (GFP). This allowed a fluorescent label (GFP) to be irreversibly and selectively expressed attached to a specific target protein, permitting the location, dynamics and function of that protein to be probed in a living cell. More recently, photoswitchable fluorescent proteins were discovered. These fluorescent proteins can be reversibly switched between fluorescent (on) and nonfluorescent (off) states by light. This property allowed the development of super-resolution fluorescence bioimaging, which improved the spatial resolution of fluorescence microscopy by more than 10 times, allowing details much smaller than the wavelength of light to be observed in living systems. However, applications of photoswitchable proteins are limited because the light used to generate fluorescence from their 'on' states also switches the proteins 'off', causing the image to fade. A higher level of control is offered by the recently discovered third generation fluorescent proteins (3G FPs), which have three states, an off-state and a switching state in thermal equilibrium with an on-state. These three states can be independently excited, so switching off is decoupled from observation from the on-state. This development will lead to enhanced super-resolution imaging, and has the potential for the development of new multicolour imaging methods. However, the mechanism connecting these states is completely unknown.Our experiments will allow us to probe in detail the effects of light on all three states of 3G FPs. We will measure the rates of the interconversion between the three states as well as the structural changes that accompany them. Since the fastest of the interconversion reactions are extremely fast (thousand-billionths of a second) we will use the tools of ultrafast laser spectroscopy to make our observations. Our ultrafast experiments probe populations of reactant, intermediate and product states through their absorption spectroscopy, while their structures are probed through ultrafast vibrational spectroscopy. These measurements will lead to a detailed picture of the photoconversion mechanism in 3G FPs. Once the mechanism is established, we will apply the tools of chemical biology to make mutations in key residues that will (i) test our ideas of the mechanism and (ii) optimise the photoswitching rate, and thus yield superior 3G FPs for bioimaging.Photoactive proteins such as FPs offer a unique opportunity to observe protein structure evolution in real time. In particular time resolved vibrational spectroscopy yields structural data from femtoseconds out to milliseconds. This information has great importance, as it can be compared with the results of computational calculations and independent experiments on protein structural dynamics. Together this will yield the most detailed insights yet into the dynamics of proteins undergoing their function, which in turn enhances our understanding of the nature of drug-protein interactions.